Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of Sporotrichosis

Abstract

Sporothrix schenckii isolates of fixed and lymphocutaneous clinical forms from Mexico (MX), Guatemala (GT), and Colombia (CO) as well as environmental isolates from MX were studied by analyzing their phenotypic characteristics (conidial length, thermotolerance by percent growth inhibition [GI] at 35 and 37 degrees C, median lethal dose [LD(50)]) and genotypic characteristics (by random amplified polymorphic DNA [RAPD] analysis-PCR). A significant difference (P < 0.01) in the mean conidial length of S. schenckii clinical isolates from CO ( = 4.03 +/- 1.04 microm) compared with those of clinical isolates from MX ( = 2.06 +/- 0.53 microm) and GT ( = 2.68 +/- 0.83 microm) was observed. The lowest thermotolerance, as determined by measurement of percent GI, was exhibited by isolates from CO at 35 degrees C ( = 50.1% +/- 15.9%) and 37 degrees C ( = 72.7% +/- 10.9%). In general, the highest virulence, as determined by measurement of the LD(50) for mice, was observed for the MX environmental isolates. RAPD analysis-PCR with 10-mer primers OPBG-01, OPBG-14, and OPBG-19 generated 52 reproducible bands. The 44 Sporothrix isolates fell into four major groups by hierarchical cluster analysis. The first group (group I), formed by 25 (of 27) isolates from MX, had two subgroups: subgroup Ia with 10 environmental isolates and subgroup Ib with 14 clinical isolates. The second group (group II) had two subgroups: subgroup IIa, formed by isolates from CO, and subgroup IIb, formed by isolates from GT. Groups III and IV each had only one clinical isolate from MX. A principal-component analysis of the same data yielded three distinct groups, depending on the geographical origins of the isolates, including the isolates in groups III and IV from MX, which were grouped with the isolates from MX by principal-component analysis. This study revealed that isolates from CO had low thermotolerances at 35 and 37 degrees C and could be associated with superficial skin lesions in patients with fixed clinical forms of sporotrichosis, the most frequent form of the disease in CO. Distinct patterns dependent on geographical origins were also revealed by RAPD analysis-PCR, but these had no relation to the clinical form of the disease.

FIG. 1.

RAPD analysis-PCR profiles of 18 isolates of S. schenckii from Mexican patients generated with primer OPBG-14. Lanes M, 123-bp molecular size marker ladder. The gel was stained with ethidium bromide. The image was inverted with GeneSnap software (Syngene) for the best resolution. Image analysis was performed as described in Material and Methods.

FIG. 2.

Relationship of S. schenckii isolates. The phenogram was generated from genetic similarity coefficients obtained by determination of the presence and absence of a total of 52 DNA bands from 44 isolates and is based on UPGMA. Dark green, clinical isolates from MX; light green, environmental isolates from MX.

FIG. 3.

PCA. The first three components explain 50% of the observed variation. Each of the three groups formed represents isolates from the three geographical regions studied (MX, GT, and CO). The group formed with isolates from MX includes clinical (C) and environmental (E) isolates.